DESCRIPTION (provided by investigator): This Small Business Innovation Research proposes to combine the best features of three different technologies: immunoassay, cell culture and real-time polymerase chain reaction (PCR), into one integrated test for environmental detection of the CDC Category A agents. Phase I will comprise development of the biosensor instrument, the test cartridge, reagents, and the assays. One bioterrorism agent, anthrax spores, will be used in Phase I. The proposed Three-Step Biosensor will have the following properties: a rapid, sensitive immunoassay that detects pathogens within 20 minutes, a cell culture step that verifies the viability of the pathogens, a real-time PCR step that confirms positive immunoassay results, quantitation of the number of organisms or toxin molecules in the sample, a compact and self-contained disposable test cartridge that safely confines the environmental sample and all test by-products, automated sample preparation and data acquisition, and a portable instrument format for use in the field by first responders The integrated test will be simultaneously more sensitive and more specific than current methods, both for on-site rapid tests and laboratory applications. By selectively capturing pathogenic anthrax spores from environmental samples and purifying them from all other organisms in the sample, the biosensor can provide a spore-derived vegetative cell lysate free of inhibitors for subsequent PCR reactions. The test addresses the root causes of both false negatives (sensitivity, viability, inhibitors) and false positives (cross-reaction, contamination) that affect other methods. Because it confirms organism viability, the test will enable real time monitoring of decontamination efforts, such as that recently performed at Washington DC postal facilities and government offices. [unreadable] [unreadable] [unreadable]